1. Field of the Invention
Polypeptides in the IpaH superfamily have been discovered to be a new class of E3 ubiquitin ligases unrelated to known E3 ubiquitin ligases such as RING. U box, and HECT-domain ligases. A ubiquitin ligase attaches the protein ubiquitin to a substrate protein. This post-translation modification affects the intracellular trafficking of the ubiquitinated substrate protein and has been shown to direct a ubiquitinated substrate protein to the proteasome for destruction. The ligase of the invention may be used to construct polypeptides which recognize a specific substrate protein and ubiquitinate it or to identify molecules which block or inhibit E3 ubiquitin ligase activity of the IpaH superfamily, such as E3 ligases expressed by the bacterial pathogens Salmonella and Shigella. 
2. Description of the Related Art
The specific and covalent addition of ubiquitin to proteins, known as ubiquitination, is a eukaryotic-specific modification central to many cellular processes, such as cell cycle progression, transcriptional regulation, and hormone signalling. Ubiquitination involves the conjugation of one or more ubiquitin moieties on to a substrate or target protein. Mono- and multi-ubiquitinations can trigger an alteration of the localization and/or activity of a target protein, while poly-ubiquitination can modulate the properties of the target protein or constitute a signal for its degradation by the 26s proteasome, Angot et al., PLOS Pathogens 3:0001 (January, 2007).
The regulated destruction of proteins via the ubiquitin proteasome pathway governs many cellular processes including cell-cycle progression and signal transduction pathways, such as the NF-κB pathway. Invading pathogens are sensed by host cells through surveillance systems that initiate signalling cascades alerting the immune system to the presence of pathogens. These signalling cascades include both MAPK and nuclear factor-κB (NF-κB) programs that induce cytokine production and ultimately result in inflammation (Inohara et al., 2005).
The process of ubiquitination requires a ubiquitin-activating enzyme (E1) which uses ATP to activate the ubiquitin protein, a limited number of ubiquitin-conjugating enzymes (E2) which receive the activated ubiquitin and can transfer an activated ubiquitin molecule to an ubuitin ligase or to a substrate protein in the presence of an ubiquitin ligase, and a large number of ubiquitin-ligase enzymes (E3) which recognize and recruit particular substrate proteins and thus control the nature and the specificity of ubiquitination.
The C-terminal Gly residue of ubiquitin is charged via a thioesther linkage onto a Cys residue of E1 and transferred to a Cys residue of E2s. E3s recruit ubiquitinated E2s to specific substrates that are ubiquitinated on Lys residues by an amide linkage. RING and U-box E3s promote the transfer of ubiquitin from E2s to targets, whereas HECT-domain E3s transfer ubiquitin onto one of their Cys residues and then to targets (Ardley and Robinson, 2005; Liu, 2004).
The ubiquitin moiety of ubiquitinated targets can then be ubiquitinated on Lys residues 48 or 63 to produce polyubiquitinated targets. Ubiquitin chains constructed by Lys-48 linkages target proteins for destruction by the proteasome whereas those constructed by Lys-63 linkages leads to altered protein function, such as the activation of kinases (Liu, 2004), or localization.
Bacteria of Shigella spp. cause shigellosis in humans by invading the colonic mucosa. Their virulence is dependent upon a 200-kb plasmid encoding a type III secretion (T3S) system (Parsot, 2005). The type III secretion (T3S) apparatus involves the injection of bacterial effector proteins into eukaryotic host cells by many gram-negative bacteria pathogenic for plants or animals, (Galan and Cossart, 2005). Shigella effector proteins that promote bacterial entry are produced and stored within the bacterium at 37° C. and transit through the T3S apparatus upon contact with epithelial cells (Menard et al., 1994). A second wave of effectors, whose functions are unknown, are produced only after contact with host cells (Demers et al., 1998). These latter effectors include nine closely related IpaH proteins that are the effectors most abundantly produced by Shigella (Demers et al., 1998). Expression of these effectors is dependent upon an AraC family member, MxiE, which activates transcription in response to the activation of the T3S apparatus (Mavris et al., 2002; Penno et al., 2005).
Defining the activity of T3S effectors is key to understanding pathogenesis, however, many effectors share little sequence similarity with proteins of known function. T3S effectors are injected into eukaryotic cells and their molecular targets are intracellular. Since yeast have many proteins and processes well conserved in higher eukaryotes, they have been used to model and gain clues as to the roles of effector proteins, such as T3S effectors (Valdivia, 2004).
Recently, studies in yeast helped to elucidate the function of the Shigella effectors IpgB1 and IpgB2 that act as G protein mimics (Alto et al., 2006). To gain insight to IpaH activity, the inventors utilized Saccharomyces cerevisiae as a surrogate model. It was found that expression of the effector molecule IpaH9.8 in yeast disrupts signalling through the pheromone response MAPK pathway by promoting the proteasome-dependent degradation of the MAPKK Ste7. In vitro assays were used to demonstrate that effectors of the IpaH superfamily, including IpaH9.8 from Shigella and SspH1 from Salmonella, constitute a novel class of E3 ubiquitin ligases.